The quantitation of bacterial viruses is important to a number of disciplines. For example, the presence of bacteriophage in a sample is one method for detecting the presence or absence of bacteria in that particular sample. The presence of these indicator organisms is widely used to assess bacterial contamination in various products. Both water and food quality are defined, in part, by the presence or absence of members of the "coliform" group, including the presence of Escherichia coli in a sample. Coliforms include members of the Enterobacteriaceae group and have the ability to ferment lactose with gas production. The genera Citrobacter, Enterobacter, Klebsiella and Escherichia are generally listed members of the coliform group.
In addition, bacteriophage are used in molecular studies for gene manipulation, as evidenced by the extensive commercial use of genetically modified bacteriophage vectors. Genetically modified bacteriophage are available from commercial suppliers including, but not limited to, Stratagene (La Jolla, Calif.), Invitrogen (San Diego, Calif.) and New England Biolabs (Beverly, Mass.). Rapid quantitation of bacteriophage is important to expediting biotechnology research.
Lytic bacterial viruses replicate within the bacterial cell resulting in bacterial lysis to release virus progeny. Lytic bacteriophages form substantially clear plaques on a lawn of bacteria (i.e., a confluent covering of bacteria). Nonlytic bacteriophages may not lyse a cell. Instead, the rate of bacterial cell growth slows and the viruses form turbid plaques on a bacterial lawn.
Standard petri-plate type assays for bacteriophage are known in the art. In these assays, agar-containing media is poured into petri-plates. A sample suspected of containing bacteriophage or known to contain bacteriophage is combined with bacteria susceptible to infection by that type of bacteriophage in a top agar-containing media. The top agar is poured over the agar plate, allowed to solidify, and the plate is incubated until areas of bacterial lysis, termed plaques, are observed on the lawn of bacteria. The plaques are counted and the number of plaques is adjusted in view of the original sample dilution to obtain the concentration of bacteriophage in the sample. Examples of these methods are disclosed, for example, in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 1989, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). Results are generally expressed as plaque forming units (pfu)/ml.
Standard petri-plate bacteriophage assays are tedious and cumbersome and often produce inaccurate results because it is often difficult to see all of the virus plaques on the bacterial lawn. Moreover, standard agar-containing petri-plates have a relatively short shelf half-life. The agar can dry out and the plates generally require refrigeration. A ready-to-use device to facilitate bacteriophage quantitation is needed.
The prior art has provided several devices that are useful for assaying liquid samples for bacteria and molds. German patent application No. 2055741, published May 19, 1971, discloses a microbiological growth medium comprised of an inert card or strip coated with a dry-gelled medium. U.S. Pat. Nos. 4,565,783, 5,443,963, 5,462,860, and 5,232,838 provide a culture media device comprising a cold-water soluble dry powder containing a gelling agent and microbial growth nutrients coated on a water-proof surface and examples of these devices are commercially available as "PETRI-FILM" devices from Minnesota Mining and Manufacturing Co., St. Paul, Minn. In these references, a transparent, read-through cover sheet is positioned on top of a surface. The application of a liquid sample to the device hydrates the gelling agent on the surface to form a gelatinous medium for growing microorganisms. These references provide flat growing surfaces with covers that contact the surface containing the organism. Such devices are not suitable for quantitating bacteriophage since direct contact of a cover with a bacterial lawn infected with bacteriophage would smear the plaques and produce inaccurate results for bacteriophage quantitation.
U.S. Pat. No. 5,089,413 discloses a device for growing microorganisms including aerobic microorganisms such as mold. The device employs an air permeable membrane to permit the growth of aerobic organisms. A spacer is disclosed in the device to define the growth region and confine an aqueous sample to the growth region of the medium. In this patent, like those cited above, the cover is designed to contact the growth surface to disperse the sample across the growth surface of the device.
There remains a need for a device and a method to detect bacteriophage quickly and easily without requiring significant preparation steps to produce and maintain a supply of solid media coated plates suitable for bacteriophage replication on a lawn of bacteria.